Reproductive biotechnology such as in vitro fertilization, the creation of transgenic animals or cloning by nuclear transfer depends on the use of fully grown, meiotically competent oocytes capable of completing meiotic maturation by reaching the stage of metaphase II. However, there exists only a limited quantity of these oocytes in the ovaries of females. In view of their limited number, growing oocytes without meiotic competence represent a possible source. The mechanisms controlling the acquisition of meiotic competence, however, are still not completely clear. A gas with a short half-life, nitric oxide (NO), produced by NO-synthase (NOS) enzyme can fulfill a regulatory role in this period. The objective of this study was to ascertain the role of NO in the growth phase of pig oocytes and its influence on the acquisition of meiotic competence with the help of NOS inhibitors, NO donors and their combinations. We demonstrated that the selective competitive iNOS inhibitor aminoguanidine and also the non-selective NOS inhibitor L-NAME block meiotic maturation of oocytes with partial or even full meiotic competence at the very beginning. NOS inhibitors influence even competent oocytes in the first stage of meiotic metaphase. However, blockage is less effective than at the beginning of meiotic maturation. The number of parthenogenetically activated competent oocytes greatly increased in a pure medium after inhibitor reversion. A large quantity of NO externally added to the in vitro cultivation environment disrupts the viability of oocytes. The effectiveness of the inhibitor can be reversed in oocytes by an NO donor in a very low concentration. However, the donor is not capable of pushing the oocytes farther than beyond the first stage of meiotic metaphase. The experiments confirmed the connection of NO with the growth period and the acquisition of meiotic competence. However, it is evident from the experiments that NO is not the only stimulus controlling the growth period.

When cultured for an extended time, pig oocytes that matured in vitro to the stage of metaphase II undergo the complex process designated as ageing. Under our conditions, some pig oocytes aged 3 days remained at the stage of metaphase II (22%), but others underwent spontaneous parthenogenetic activation (45%), and still others perished through fragmentation (28%) or lysis (5%). Activation of protein kinases C (PKCs) using phorbol-12-myristate-13-acetate (PMA) protects oocytes from fragmentation. None of the oocytes were fragmented after 3 days of aging in 50 nM of PMA. A similar effect (8% of fragmented oocytes) was observed after a 3-day treatment of aging oocytes with 100 mM of 1-stearoyl-2arachidonoyl-sn-glycerol (STEAR). PMA and STEAR activate both calcium-dependent and calcium-independent PKCs. This combined effect on PKCs seems to be essential for the protection of oocytes from fragmentation. Neither the specific activator of calcium-dependent PKCs 1-oleoyl-2-acetyl-sn-glycerol (OLE) nor the specific activator of calcium-independent PKCs dipalmitoyl-L-a-phosphatidylinositol-3,4,5-triphosphate heptaammonium salt (DIPALM) suppressed the fragmentation of aging pig oocytes. Twenty-one percentage of oocytes fragmented when aged for 3 days in 10 mM OLE and 26% of aged oocytes fragmented in 100 nM of DIPALM. However, fragmentation was significantly suppressed to 7% when the oocytes were exposed to the combination of both 10 mM OLE and 100 nM DIPALM. Aging pig oocytes cultured for 1 day with PMA maintained a high capability of being parthenogenetically activated (86% of activated oocytes), using calcium ionophore with 6-dimethylaminopurine. Ageing oocytes treated with PMA also had high capability of cleavage (82%) after their artificial parthenogenetic activation. However, their ability to develop to the stage of blastocyst (12%) was suppressed when compared with oocytes activated immediately after their maturation (29%).

Pig oocytes matured in vitro were parthenogenetically activated using nitric oxide donor SNAP (2mM). Continuous treatment successfully activated the oocytes only after more than 12 hours of exposure. Pulsatile treatments during which oocytes were repeatedly exposed to 2mM SNAP for a short time (10, 20 or 30 minutes) were more efficient with regard to the activation rate, even when the total exposure time did not exceed 4 hours. Parthenogenetic development was very limited after continuous treatment with 2mM SNAP. A significantly higher proportion of developing parthenogenetic embryos was observed after the pulsatile treatment (development to the morula stage 0 vs. 18%; development to the blastocyst 0 vs. 7%; P < 0.05). However, this developmental rate was significantly lower (P < 0.05) than the development induced by conventional activation treatment with calcium ionophore (development to the morula stage, 23%; development to the blastocyst stage, 18%). When we combined pulsatile SNAP-treatment with the effect of protein kinase inhibitor 6-dimethyl aminopurine (6-DMAP) (2mM 6-DMAP for 2 hours) or with the inhibitor of protein synthesis cycloheximide (CHX) (10 µM CHX for 2 hours), we observed a significant increase (P < 0.05) in the activation rate when compared to the respective pulsatile SNAP-treatment without 6-DMAP or CHX (63 vs. 78% of activated oocytes for 6-DMAP; 63 vs. 83% of activated oocytes for CHX). However, the development of parthenogenetic embryos was not enhanced when the pulsatile SNAP-treatment was combined with 6-DMAP or with CHX.